Protein Isolation and PAGE Electrophoresis

By Amanda M. Icazatti-Burtell

The body produces a certain amount of metabolites that are required for physiology and functioning. Many of these metabolites are proteins which act as catalysts in chemical reactions. If there are any damages to these proteins or enzymes, our body can be affected by its deficiency or malfunctioning.

Plasminogen activator (PA), an enzyme within our body, which activates other enzymes to prevent clotting in the bloodstream, is being isolated from Hela cells using magnetic nanoparticles. Magnetic nanoparticles are being used because they are easy to separate from other suspended solids, can be easy automated, present affinity binding, and make the process much faster.

Chromatography and Zymography are important processes in determining the presence of a protein and the enzyme activity, respectively. In chromatography, components of a mixture are separated or purified in order to measure the relative proportions of the substance in study. It is known that proteins have an absorbance to a specific wavelength of 280nm and by using chromatography the substrate obtained can reveal the presence or absence of a protein. Moreover, zymography supports the purification process, since it is through this technique that can be determined if the substrate under study has any enzymatic activity.

Polyacrylamide gel electrophoresis (PAGE) was performed to obtain a separation of proteins by size isolated in the experiment. This process is very useful for the analysis of proteins and can help determined their magnitude.

During the course of the experiment a reactant used seem to be contaminated, because the absorbency results obtained for the elute buffer were too high at a stage in which the absorbency had to be much lower.

Biochemistry is a key to understanding how the biological world and the chemical world support and complement each other.